Detection of IL35+ Exosomes as a Marker for Peripheral Tolerance

检测 IL35 外泌体作为外周耐受性的标志物

基本信息

批准号：
9978316
负责人：
Jeremy A Sullivan
金额：
$ 7.75万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2021-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9978316
关键词：
Address Animals Antibodies Antigens Ascites Autoantigens Autoimmune Process Biological Assay Blood CD81 gene Cell Culture Techniques Cells Control Animal Culture Media Data Delayed Hypersensitivity Detection Effector Cell Engineering Enzyme-Linked Immunosorbent Assay Failure Family Future Hour Human Human Herpesvirus 4 Immune Immune response Immunize Immunology Immunosuppression Inflammatory Response Interleukin-12 Interleukins Knockout Mice Label Lead Liquid substance LoxP-flanked allele Lymph Lymphocyte Malignant Neoplasms Measures Mediating Membrane Mus Physiological Production Property Protein Subunits Proteins Protocols documentation Regulation Regulatory T-Lymphocyte Reporter Reporting SCID Mice Serum Swelling TNFSF5 gene Testing Tetanus Toxoid Therapeutic Transfusion Transmission Electron Microscopy Transplant Recipients Transplantation cell transformation cytokine design exosome extracellular vesicles member mouse model neutralizing antibody nonhuman primate peripheral tolerance response transplant model

项目摘要

ABSTRACT Interleukin 35 (IL35) has emerged as a potent immuno-suppressive cytokine in cancer, auto-immune and transplant immunology. A member of the IL12 family of cytokines, IL35 is a heterodimeric cytokine composed of the protein subunits Epstein Barr Virus Induced 3 (Ebi3) and p35 and is produced and secreted predominantly by regulatory T cells (Tregs). While IL35 has been implicated in the regulation of a number of cellular immune responses, there still exists significant debate as to whether the cytokine actually exists. This debate is predicated on the fact that attempts to isolate IL35 from blood, physiological solutions like ascites or lymph, or even cell culture supernatants have failed. We have recently immune-precipitated both Ebi3 and p35 with an antibody to the tetraspanin CD81 from mouse lymphocytes. Tetraspanins are membrane spanning proteins associated with the formation of the small, extracellular vesicles, exosomes. This interesting observation prompted us to examine whether IL35 exists as Ebi3 and p35 proteins associated with tetraspanins and secreted and acquired as an exosome. To test the idea that IL35 exists as an exosome dependent cytokine, we designed the following specific aims: Specific Aim 1: To test whether exosomes isolated from serum of tolerized groups of tetraspanin knockout mice (CD81, CD63, CD9) lose Ebi3 and p35 positive exosomes. Specific Aim 2: To examine whether lymphocytes from tolerized-CD81, CD63 or CD9 knockout mice produce and release functional IL35+ immuno-suppressive exosomes. The successful completion of the outlined specific aims in this proposal will fill a significant gap in our understanding of the cytokine IL35, and will ideally lead to future applications that further our understanding of the therapeutic implications of IL35 for humans.

抽象的白细胞介素 35 (IL35) 已成为癌症、自身免疫性疾病和癌症中有效的免疫抑制细胞因子。移植免疫学。 IL35 是细胞因子 IL12 家族的成员，是一种异二聚体细胞因子，由蛋白质亚基 Epstein Barr 病毒诱导 3 (Ebi3) 和 p35 的产生和分泌主要由调节性 T 细胞 (Treg) 组成。虽然 IL35 与许多细胞因子的调节有关尽管细胞免疫反应中存在细胞因子，但对于细胞因子是否确实存在仍存在重大争论。这争论的基础是尝试从血液、腹水等生理溶液中分离 IL35 淋巴液，甚至细胞培养上清液都失败了。我们最近对 Ebi3 和 p35 进行了免疫沉淀含有来自小鼠淋巴细胞的四跨膜蛋白 CD81 抗体。四跨膜蛋白是跨膜的与小细胞外囊泡、外泌体的形成相关的蛋白质。这个有趣的观察促使我们检查 IL35 是否以 Ebi3 和 p35 蛋白的形式存在四跨膜蛋白并作为外泌体分泌和获得。检验 IL35 作为外泌体存在的观点依赖细胞因子，我们设计了以下具体目标：具体目标1：测试外泌体是否从四跨膜蛋白敲除小鼠（CD81、CD63、CD9）耐受组的血清中分离出来，失去了 Ebi3 和 p35 阳性外泌体。具体目标 2：检查淋巴细胞是否来自耐受 CD81、CD63 或 CD9 基因敲除小鼠产生并释放功能性 IL35+ 免疫抑制外泌体。成功者完成本提案中概述的具体目标将填补我们对细胞因子 IL35，理想情况下将导致未来的应用，进一步加深我们对治疗的理解 IL35 对人类的影响。